Fast and reliable methods for investigating drug actions on intracellular chemistry are in high demand today. Such protocols could include screening for ligands and substrates that interact with organelle-bound receptors and cytosolic enzymes, respectively. Also methods that allow for characterization and detection of all proteins inside cells would be extremely valuable, not the least in the area of proteomics. Highly specific enzymes substrates and protein probes are available that makes it possible to detect particular components in cells. (Tsien R Y, Annu. Rev. Biochem. 1998, 67, 509-44). For example, there are a variety of substrates available that can be employed as light switches in the substrate-product conversion step. Also, specific protein-protein interactions can be identified by the use of a fluorescence indicator coupled to protein splicing (Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y, Anal. Chem. 2000, 72, 5151-57). Although these probes and indicators are available, the main challenge so far in applying these probes and indicators is to introduce them into the cellular interior. Many of these probes and indicators as well as many other compounds for biological and medical use including drugs and biomolecules that are of interest to include in cells are polar. Polar solutes are cell-impermeant and unable to pass biological membranes.
Furthermore, methods that allow for the detection of DNA-protein, protein-protein and many more interactions inside cells would be a valuable tool in many areas. Likewise, the ability to introduce viruses, bacteria, antibodies, and colloidal particles to cells is judged to be of importance in many areas of the biosciences. It is, however, difficult to transfer all these compounds and particles to the cytosol of a cell owing to the presence of a cell plasma membrane barrier, which acts as a physical boundary to the external solution that prevents the entrance of exogenous compounds and particles.
It has for a long time been recognized that cell membranes can be permeabilized by pulsed electric fields (see e.g. Zimmermann, U. Biochim. Biophys Acta, 694, 227-277 (1982). This technique is called electroporation. It is known from work on electrochemical detection in capillary electrophoresis (CE) that the voltage applied over an electrolyte filled capillary (EFC) will create an electric field at the capillary outlet (Lu, W.; Cassidy, M. Anal. Chem. 1994, 66, 200-204). This electric field at the tip of an EFC working against ground potential can be used for electroporation. The same EFC that performs the electroporation also delivers the agents of interest to the cell. It can be shown that the magnitude of the electric field along the axis of symmetry of the EFC lumen, extending out into solution is given by:
                              E          ⁡                      (                          Z              ,              Ψ                        )                          =                              Ψ                          L              c                                [                                    Z                                                [                                      1                    +                                                                  (                        Z                        )                                            2                                                        ]                                                  1                  2                                                      -            1                    ]                                    (        1        )            Where Z is the dimensionless distance from the tip of the EFC, z/a, where z is distance from the EFC tip and a is the EFC lumen radius. Ψ is the applied potential in volts and Lc is the length of the EFC. This equation can be integrated to find the potential drop along the cylindrical axis outside the capillary.
                              V          ⁡                      (            Z            )                          =                                            a              ⁢                                                          ⁢              Ψ                                      L              c                                ⁢                      (                                                            (                                                            Z                      2                                        +                    1                                    )                                                  1                  /                  2                                            -              Z                        )                                              (        2        )            The transmembrane voltage can thus be approximated from this field by using equation (2) as described above.
The inventors have previously demonstrated the concept of electroporation using a singular EFC containing a homogeneous electrolyte solution (K. Nolkrantz, R. I. D. Karlsson, C. Farre, A. Brederlau, C. Brennan, P. S. Eriksson, S. G. Weber, M. Sandberg, O. Orwar Anal. Chem., (2001) 73, 4469-4477; WO 9924110).